Mesoporous nitric oxide-releasing silica particles, methods of making, and uses thereof

ABSTRACT

Nitric oxide-releasing materials, methods of making nitric oxide-releasing materials, and uses of nitric oxide-releasing materials are provided. The nitric oxide-releasing materials include a mesoporous silica core and an outer surface having a plurality of nitric oxide donors. In an exemplary aspects, the nitric oxide-releasing material includes a mesoporous diatomaceous earth core, and an outer surface having a plurality of S-nitroso-N-acetyl-penicillamine groups covalently attached thereto. Uses of the nitric oxide-releasing materials can include coatings for medical devices such as catheters, grafts, and stents; wound gauzes; acne medications; and antiseptic mouthwashes; among others.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.provisional application entitled “MESOPOROUS NITRIC OXIDE-RELEASINGSILICA PARTICLES, METHODS OF MAKING, AND USES THEREOF” having Ser. No.62/539,788, filed Aug. 1, 2017, the contents of which are incorporatedby reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under awards K25HL111213and R01 HL134899 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The present disclosure generally relates to nitric oxide donors.

BACKGROUND

Nitric oxide (NO), a gaseous diatomic free radical endogenously producedvia the sequential enzymatic oxidation of L-arginine, plays an importantand far-reaching role in human physiology [1, 2]. In the 30 years sinceit was first identified as endothelium-derived relaxing factor, NO hasbeen shown to modulate a number of biological processes such as smoothmuscle relaxation, cell proliferation, vasodilation, neurotransmission,cell signaling, the inhibition of platelet adhesion and aggregation, andimmune system regulation [1, 3-5]. Nitric Oxide's efficacy in thesediverse roles stems from its high membrane diffusivity and excellentreactivity with a variety of chemical species including oxygen,superoxide anions, oxyhemoglobin, thiols, pyrimidine bases, lipids, andmetallic complexes [1, 6, 7]. In recent decades, “donor” molecules thatrelease NO at or above physiological levels have been incorporated intoa number of biomaterials in order to artificially induce therapeuticeffects consistent with those of endogenous NO [4-6, 8-10]. These donormolecules act as NO delivery vehicles, making targeted NO administrationfeasible by eliminating the spatial and temporal issues surrounding themolecule's short physiological half-life and diffusion distance (1-3 μsand 100-200 μm, respectively) [6, 7, 9, 11, 12].

S-Nitrosothiols (RSNOs) in particular have emerged as one of the mostpopular classes of NO donors [1, 9, 10]. Synthesized via an acidifiednitrosation reaction between thiols (RSH) and nitrite, nitrogen oxides,or alkyl nitrites, RSNOs undergo chemical, photolytic, and thermaldecomposition to release NO [9, 13]. S-nitroso-N-acetyl-penicillamine(SNAP), the nitrosated form of N-acetyl-penicillamine (NAP), an aminoacid derivative that has been used to treat cystinuria at doses of 2-4g/day for 155 days with minimal side-effects, is one of the mostprevalent RSNO molecules due to its relatively high molecular stabilityand non-toxic origins [1, 9, 10, 14].

To date, the majority of research in the field of NO technology hasfocused on the development of antithrombogenic and antimicrobialbiomaterials such as catheters, extracorporeal circuitry, biosensors,and biomedical device coatings [12, 15-21]. In addition, NO has alsobeen incorporated into food packaging, acne medications, wound healingmaterials, and toothpastes [22-25]. The addition of NO donor moleculesto these wide-ranging materials is accomplished by either physical(blending or swelling) or chemical means (covalent attachment) [13, 15,17, 18, 26-29]. Although easier to produce, blended and swollen NOreleasing materials often suffer from molecular leaching and diminishedrelease times [12, 13]. The covalent attachment of NO donors to polymerbackbones and release scaffolds protects against these limitations,increasing the stability, safety, and application range of materials[12, 13, 30].

A popular material choice for covalently formed NO releasing scaffoldsis silicon dioxide due to its chemical inertness, tunable particle size,affordability, and abundance [30-35]. A handful of research groups haspreviously produced silica-based NO scaffolds using a variety ofmethodologies [30-35]. For instance, Zhang et al. modified fumed silicawith amine-containing silylation reagents to create nonporousdiazeniumdiolated silica particles (0.2-0.3 μm) that enhancedthromboresistance when embedded in ECC tubing [31]. In a similarfashion, Frost et al. employed silyation agents to tether RSNOs tononporous fumed silica particles (7-10 nm) and analyzed their releasekinetics under various conditions [30]. Shin et al. synthesizednonporous diazeniumdiolated NO releasing silica particles (20-500 nm) denovo via the co-condensation of two silicon alkoxide precursors [32,33]. Hetrick et al. later tested these particles for anti-biofilm andbactericidal efficacy [36, 37]. Most recently, Soto et al. createddiazeniumdiolated porous silica particles (30-1100 nm) using a modifiedalkoxysilane co-condensation synthesis [34].

Previously synthesized scaffolds have struggled to strike an idealbalance between NO release kinetics, morphology, particle size, and easeof synthesis. Specifically, diazeniumdiolate based silica scaffoldsundergo burst release, eliminating all stored nitric oxide within hoursand limiting their utility [31-34]. Additionally, virtually allpreviously reported NO releasing scaffolds have been nonporous and onthe nanoscale [30-35]. Mesoporous, micron scale silica RSNO scaffoldshave not yet been created.

There remains a need for improved NO donors that overcome theaforementioned deficiencies.

SUMMARY

Nitric oxide donor materials, methods of making nitrogen oxide donormaterials, and various articles incorporating the nitrogen oxide donormaterials are provided that overcome one or more of the aforementioneddeficiencies. In an exemplary aspect, a nitric oxide-releasing materialis provided having a mesoporous diatomaceous earth core, and an outersurface having a plurality of S-nitroso-N-acetyl-penicillamine groupscovalently attached thereto. The materials can be useful, for instance,in medical devices such as a urinary catheter, a vascular catheter, agraft, or a stent.

In some aspects, a nitric oxide-releasing material is provided having amesoporous silica core, and an outer surface having a plurality ofmoieties having a structure according to the following formula

where A is a nitric oxide donor; where R¹ is none, a substituted orunsubstituted C₁-C₂₀ alkyl, a substituted or unsubstituted C₁-C₂₀heteroalkyl, a substituted or unsubstituted C₂-C₂₀ alkenyl, asubstituted or unsubstituted C₂-C₂₀ herteroalkenyl, a substituted orunsubstituted C₁-C₂₀ alkoxy, or a substituted or unsubstituted C₁-C₂₀heteroalkoxy; where each occurrence of R² is independently a substitutedor unsubstituted C₁-C₂₀ alkyl, a substituted or unsubstituted C₁-C₂₀heteroalkyl, a substituted or unsubstituted C₂-C₂₀ alkenyl, asubstituted or unsubstituted C₂-C₂₀ herteroalkenyl, a substituted orunsubstituted C₁-C₂₀ alkoxy, a substituted or unsubstituted C₁-C₂₀heteroalkoxy, or a bond to an oxygen atom on the outer surface so longas at least one occurrence of R² is a bond to an oxygen atom on theouter surface.

In some aspects, A is an S-nitrosothiol such asS-nitroso-N-acetyl-penicillamine, S-nitroso-N-acetyl cysteine,S-nitroso-N-acetyl cysteamine, S-nitrosoglutathione, methylS-nitrosothioglycolate, or a derivative thereof. In some aspects, thenitric oxide donor is a diazeniumdiolate such as diazeniumdiolateddibutylhexanediamine or a derivative thereof.

In some aspects. A has a structure according to the formula R⁴SNO, whereR⁴ is an amino acid or fragment thereof.

In some aspects, in the formula above, R¹ is a substituted orunsubstituted C₁-C₁₂ alkyl or a substituted or unsubstituted C₁-C₁₂aminoalkyl. In some aspects, in the formula above, each occurrence of R²is independently a substituted or unsubstituted C₁-C₆ alkyl or a bond toan oxygen atom on the outer surface.

In some aspects, a nitric oxide-releasing material provided herein has anitric oxide content of about 0.025 μmol to about 0.05 μmol per mg ofthe nitric oxide-releasing material. In some aspects, a nitricoxide-releasing material provided herein has a half-life for nitricoxide release of about 20 hours to about 40 hours. In some aspects, anitric oxide-releasing material provided herein an average pore size ofabout 300 nm to about 600 nm. In some aspects, a nitric oxide-releasingmaterial provided herein is a particle having a longest dimension ofabout 10 μm to about 20 μm. In some aspects, a nitric oxide-releasingmaterial provided herein includes a mesoporous silica core is selectedfrom the group consisting of a diatomaceous earth, a rice husk, an SAB-3type mesoporous silica, an HMS type mesoporous silica, MSU-X typemesoporous silica, an SBA-12 type mesoporous silica, an SBA-15 typemesoporous silica, an SBA-16 type mesoporous silica, and an MCM-41 typemesoporous silica.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1 is a schematic of an exemplary derivatization of nitricoxide-releasing diatomaceous earth using (3-Aminopropyl)triethoxysilaneas a representative silane.

FIG. 2 show traditional structural representations of silylation agents(upper) and conformational representations of silylation agents (lower)during attachment to silica surfaces.

FIG. 3 is a schematic representation of aminosilane attachment andself-polymerization occurring on the diatomaceous earth silica surface.

FIG. 4 shows Fourier-transform infrared spectra of unmodifieddiatomaceous earth (upper curve) and N-acetyl-penicillamine modifieddiatomaceous earth (lower curve).

FIG. 5 shows a representative 24-hour nitric oxide release profile forS-nitroso-N-acetyl-penicillamine modified diatomaceous earth (SNAP-DE)as both an instantaneous parts per billion per mg SNAP-DE (PPB/mg) valueand cumulative percentage. Because SNAP-DE nitric oxide-release levelsplateaued quickly, this profile was prepared by linearly interpolatingbetween steady-state data recorded at the beginning and end of the24-hour period.

FIGS. 6A-6B show scanning electron microscope images of diatomaceousearth (FIG. 6A) before and (FIG. 6B) after covalentS-nitroso-N-acetyl-penicillamine attachment.

FIGS. 7A-7F show energy dispersive X-ray spectra of (FIG. 7A) unmodifieddiatomaceous earth and (FIG. 7B) S-nitroso-N-acetyl-penicillaminemodified diatomaceous earth (SNAP-DE); (FIGS. 7C-7F) scanning electronmicroscope image of SNAP-DE without (7C, 7E) and with elemental sulfurmapping overlay (7D, 7F), respectively.

FIGS. 8A-8B show a graphical comparison of the viable bacterial colonyforming units per mg (CFU/mg) for unmodified diatomaceous earth andSNAP-diatomaceous earth after 24 hours of exposure. Bacteria grownwithout exposure to either diatomaceous earth orS-nitroso-N-acetyl-penicillamine modified diatomaceous earth (SNAP-DE)were used as a control. As a proof of concept, gram positive S. aureus,one of the major causal agents of biofilm formation and nosocomialinfections, was used to test the antibacterial property of SNAP-DE. Thebactericidal nature of nitric oxide killed the bacteria on a logarithmicscale (FIG. 8A) and resulted in 92.95±2.6% of reduction (FIG. 8B).

FIG. 9 shows the cytotoxic potential of S-nitroso-N-acetyl-penicillaminemodified diatomaceous earth (SNAP-DE) leachates in solution and wastested on 3T3 mouse fibroblast cells using a WST-8 dye based CCK-8 kit.Cells exposed to the leachates from diatomaceous earth and SNAP-DEdemonstrated cell viabilities similar to those of control cells notexposed to leachates.

DETAILED DESCRIPTION

In various aspects, nitric oxide-releasing materials are provided.Methods of making and uses of the nitric oxide-releasing materials arealso provided.

Diatomaceous earth (DE) consists of the fossilized 10-150 μm shells ofdiatoms, a class of unicellular marine algae possessing extraordinarilyintricate and porous three-dimensional morphologies [35, 38, 39]. Withan estimated world reserve of 800 million metric tons and countlessapplications across the food, cosmetic, chemical, pharmaceutical, andmedical industries, diatomaceous earth is a material as ubiquitous as itis versatile [35, 38]. The unique structure of diatoms, coupled withtheir high amorphous silicon dioxide content (at times 95%), renderdiatomaceous earth a low density, high surface area, chemically inert,all-natural, abrasive absorptive [35, 38]. Because of these properties,diatomaceous earth is routinely used as a filtration aid, naturaldetoxifier, cosmetic and personal hygiene abrasive, insecticide, drugdelivery and tissue engineering scaffold, wound healing agent, andpolymeric filler [35, 38, 40-43]. Chemically modifying diatomaceousearth to release nitric oxide stands to enhance the material's alreadymanifest versatility and efficacy in these biomedical applications andothers.

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. The skilled artisan will recognize many variants andadaptations of the embodiments described herein. These variants andadaptations are intended to be included in the teachings of thisdisclosure and to be encompassed by the claims herein.

All publications and patents cited in this specification are cited todisclose and describe the methods and/or materials in connection withwhich the publications are cited. All such publications and patents areherein incorporated by references as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. Such incorporation by reference is expressly limited tothe methods and/or materials described in the cited publications andpatents and does not extend to any lexicographical definitions from thecited publications and patents. Any lexicographical definition in thepublications and patents cited that is not also expressly repeated inthe instant specification should not be treated as such and should notbe read as defining any terms appearing in the accompanying claims. Thecitation of any publication is for its disclosure prior to the filingdate and should not be construed as an admission that the presentdisclosure is not entitled to antedate such publication by virtue ofprior disclosure. Further, the dates of publication provided could bedifferent from the actual publication dates that may need to beindependently confirmed.

Although any methods and materials similar or equivalent to thosedescribed herein can also be used in the practice or testing of thepresent disclosure, the preferred methods and materials are nowdescribed. Functions or constructions well-known in the art may not bedescribed in detail for brevity and/or clarity. Embodiments of thepresent disclosure will employ, unless otherwise indicated, techniquesof nanotechnology, organic chemistry, material science and engineeringand the like, which are within the skill of the art. Such techniques areexplained fully in the literature.

It should be noted that ratios, concentrations, amounts, and othernumerical data can be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a numerical range of “about 0.1%to about 5%” should be interpreted to include not only the explicitlyrecited values of about 0.1% to about 5%, but also include individualvalues (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%,2.2%, 3.3%, and 4.4%) within the indicated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, e.g. thephrase “x to y” includes the range from ‘x’ to ‘y’ as well as the rangegreater than ‘x’ and less than ‘y’. The range can also be expressed asan upper limit, e.g. ‘about x, y, z, or less' and should be interpretedto include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ aswell as the ranges of ‘less than x’, less than y’, and ‘less than z’.Likewise, the phrase ‘about x, y, z, or greater’ should be interpretedto include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ aswell as the ranges of ‘greater than x’, greater than y’, and ‘greaterthan z’. In some embodiments, the term “about” can include traditionalrounding according to significant figures of the numerical value. Inaddition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numericalvalues, includes “about ‘x’ to about ‘y’”.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. It will be further understoodthat terms, such as those defined in commonly used dictionaries, shouldbe interpreted as having a meaning that is consistent with their meaningin the context of the specification and relevant art and should not beinterpreted in an idealized or overly formal sense unless expresslydefined herein.

The articles “a” and “an,” as used herein, mean one or more when appliedto any feature in embodiments of the present invention described in thespecification and claims. The use of “a” and “an” does not limit themeaning to a single feature unless such a limit is specifically stated.The article “the” preceding singular or plural nouns or noun phrasesdenotes a particular specified feature or particular specified featuresand may have a singular or plural connotation depending upon the contextin which it is used.

The term “reactive coupling group”, as used herein, refers to anychemical functional group capable of reacting with a second functionalgroup to form a covalent bond. The selection of reactive coupling groupsis within the ability of the skilled artisan. Examples of reactivecoupling groups can include primary amines (—NH₂) and amine-reactivelinking groups such as isothiocyanates, isocyanates, acyl azides, NHSesters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes,carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, andfluorophenyl esters. Most of these conjugate to amines by eitheracylation or alkylation. Examples of reactive coupling groups caninclude aldehydes (—COH) and aldehyde reactive linking groups such ashydrazides, alkoxyamines, and primary amines. Examples of reactivecoupling groups can include thiol groups (—SH) and sulfhydryl reactivegroups such as maleimides, haloacetyls, and pyridyl disulfides. Examplesof reactive coupling groups can include photoreactive coupling groupssuch as aryl azides or diazirines. The coupling reaction may include theuse of a catalyst, heat, pH buffers, light, or a combination thereof.

The term “protective group”, as used herein, refers to a functionalgroup that can be added to and/or substituted for another desiredfunctional group to protect the desired functional group from certainreaction conditions and selectively removed and/or replaced to deprotector expose the desired functional group. Protective groups are known tothe skilled artisan. Suitable protective groups may include thosedescribed in Greene, T. W. and Wuts, P. G. M., Protective Groups inOrganic Synthesis, (1991). Acid sensitive protective groups includedimethoxytrityl (DMT), tert-butylcarbamate (tBoc) and trifluoroacetyl(tFA). Base sensitive protective groups include9-fluorenylmethoxycarbonyl (Fmoc), isobutyrl (iBu), benzoyl (Bz) andphenoxyacetyl (pac). Other protective groups include acetamidomethyl,acetyl, tert-amyloxycarbonyl, benzyl, benzyloxycarbonyl,2-(4-biphεnylyl)-2-propyloxycarbonyl, 2-bromobenzyloxycarbonyl,tert-butyl₇ tert-butyloxycarbonyl,I-carbobenzoxamido-2,2.2-trifluoroethyl, 2,6-dichlorobenzyl,2-(3,5-dimethoxyphenyl)-2-propyloxycarbonyl, 2,4-dinitrophenyl,dithiasuccinyl, formyl, 4-methoxybenzenesulfonyl, 4-methoxybenzyl,4-methylbenzyl, o-nitrophenylsulfenyl, 2-phenyl-2-propyloxycarbonyl,α-2,4,5-tetramethylbenzyloxycarbonyl, p-toluenesulfonyl, xanthenyl,benzyl ester, N-hydroxysuccinimide ester, p-nitrobenzyl ester,p-nitrophenyl ester, phenyl ester, p-nitrocarbonate,p-nitrobenzylcarbonate, trimethylsilyl and pentachlorophenyl ester.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In some embodiments, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chains,C₃-C₃₀ for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer.Likewise, in some embodiments cycloalkyls have from 3-10 carbon atoms intheir ring structure, e.g. have 5, 6 or 7 carbons in the ring structure.The term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having one or more substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents include, but are not limited to, halogen, hydroxyl,carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oran aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, or from one to six carbon atoms in its backbonestructure. Likewise, “lower alkenyl” and “lower alkynyl” have similarchain lengths. Throughout the application, preferred alkyl groups arelower alkyls. In some embodiments, a substituent designated herein asalkyl is a lower alkyl.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Cycloalkyls can besubstituted in the same manner.

The term “heteroalkyl”, as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In some embodiments, the “alkylthio”moiety is represented by one of —S-alkyl, —S-alkenyl, and —S-alkynyl.Representative alkylthio groups include methylthio, and ethylthio. Theterm “alkylthio” also encompasses cycloalkyl groups, alkene andcycloalkene groups, and alkyne groups. “Arylthio” refers to aryl orheteroaryl groups. Alkylthio groups can be substituted as defined abovefor alkyl groups.

The terms “alkenyl” and “alkynyl”, refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy, andtert-butoxy. An “ether” is two hydrocarbons covalently linked by anoxygen. Accordingly, the substituent of an alkyl that renders that alkylan ether is or resembles an alkoxyl, such as can be represented by oneof —O-alkyl, —O-alkenyl, and —O-alkynyl. Aroxy can be represented by—O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as definedbelow. The alkoxy and aroxy groups can be substituted as described abovefor alkyl.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀, and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈ or R₉ and R₁₀ taken together with the Natom to which they are attached complete a heterocycle having from 4 to8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In some embodiments, only one of R₉ or R₁₀ canbe a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not form animide. In still other embodiments, the term “amine” does not encompassamides, e.g., wherein one of R₉ and R₁₀ represents a carbonyl. Inadditional embodiments, R₉ and R₁₀ (and optionally R′₁₀) eachindependently represent a hydrogen, an alkyl or cycloakly, an alkenyl orcycloalkenyl, or alkynyl. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted (asdescribed above for alkyl) or unsubstituted alkyl attached thereto,i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉ and R₁₀ are as defined above.

“Aryl”, as used herein, refers to C₅-C₁₀-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring systems. Broadly defined, “aryl”, as used herein,includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example, benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics”. The aromaticring can be substituted at one or more ring positions with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (orquaternized amino), nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN; and combinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl”.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

“Heterocycle” or “heterocyclic”, as used herein, refers to a cyclicradical attached via a ring carbon or nitrogen of a monocyclic orbicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ringatoms, consisting of carbon and one to four heteroatoms each selectedfrom the group consisting of non-peroxide oxygen, sulfur, and N(Y)wherein Y is absent or is H, O, (C₁-C₁₀) alkyl, phenyl or benzyl, andoptionally containing 1-3 double bonds and optionally substituted withone or more substituents. Examples of heterocyclic ring include, but arenot limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Heterocyclicgroups can optionally be substituted with one or more substituents atone or more positions as defined above for alkyl and aryl, for example,halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, and—CN.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, ancycloalkenyl, or an alkynyl, R′₁₁ represents a hydrogen, an alkyl, acycloalkyl, an alkenyl, an cycloalkenyl, or an alkynyl. Where X is anoxygen and R₁₁ or R′₁₁ is not hydrogen, the formula represents an“ester”. Where X is an oxygen and R₁₁ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R₁₁ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen and R′₁₁ is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R′₁₁ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “monoester” as used herein refers to an analogue of adicarboxylic acid wherein one of the carboxylic acids is functionalizedas an ester and the other carboxylic acid is a free carboxylic acid orsalt of a carboxylic acid. Examples of monoesters include, but are notlimited to, to monoesters of succinic acid, glutaric acid, adipic acid,suberic acid, sebacic acid, azelaic acid, oxalic and maleic acid.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Examples of heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium. Other heteroatoms includesilicon and arsenic.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds described herein. In the broadest sense,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, preferably 1-14 carbonatoms, and optionally include one or more heteroatoms such as oxygen,sulfur, or nitrogen grouping in linear, branched, or cyclic structuralformats. Representative substituents include alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.

Heteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. It is understood that“substitution” or “substituted” includes the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, i.e. a compound that does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

In a broad aspect, the permissible substituents include acyclic andcyclic, branched and unbranched, carbocyclic and heterocyclic, aromaticand nonaromatic substituents of organic compounds. Illustrativesubstituents include, for example, those described herein. Thepermissible substituents can be one or more and the same or differentfor appropriate organic compounds. The heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms.

In various aspects, the substituent is selected from alkoxy, aryloxy,alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate,carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl,heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide,sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each ofwhich optionally is substituted with one or more suitable substituents.In some embodiments, the substituent is selected from alkoxy, aryloxy,alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate,carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl,heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonicacid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy,alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate,carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl,heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonicacid, sulfonamide, and thioketone can be further substituted with one ormore suitable substituents.

Examples of substituents include, but are not limited to, halogen,azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, thioketone, ester, heterocyclyl, —CN, aryl, aryloxy,perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl,heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters,carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl,alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl,carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl,alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl,perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, thesubstituent is selected from cyano, halogen, hydroxyl, and nitro.

The term “copolymer” as used herein, generally refers to a singlepolymeric material that is comprised of two or more different monomers.The copolymer can be of any form, such as random, block, graft, etc. Thecopolymers can have any end-group, including capped or acid end groups.

The terms “mean particle size” and “average particle size,” as usedinterchangeably herein, generally refer to the statistical mean particlesize (diameter) of the particles in the composition.

The terms “mean pore size” and “average pore size,” as usedinterchangeably herein, generally refer to the statistical mean poresize (diameter) of the pores in a porous material.

The terms “monodisperse” and “homogeneous size distribution”, as usedinterchangeably herein, describe a population of particles or pores allhaving the same or nearly the same size. As used herein, a monodispersedistribution refers to distributions in which 90% of the particles orpores in the distribution have a size that lies within 5% of the meansize for the distribution.

As used herein, the term “linker” refers to a carbon chain that cancontain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long. Linkersmay be substituted with various substituents including, but not limitedto, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino,dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl,heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylicacid, ester, thioether, alkylthioether, thiol, and ureido groups. Thoseof skill in the art will recognize that each of these groups may in turnbe substituted. Examples of linkers include, but are not limited to,pH-sensitive linkers, protease cleavable peptide linkers, nucleasesensitive nucleic acid linkers, lipase sensitive lipid linkers,glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers,photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers(e.g., esterase cleavable linker), ultrasound-sensitive linkers, andx-ray cleavable linkers.

Nitric Oxide-Releasing Materials

A variety of nitric oxide-releasing materials are provided. The nitricoxide-releasing materials with mesoporous structures can provide forcontrollable and sustained release of nitric oxide, even in biologicalenvironments. An exemplary nitric oxide-releasing material includes amesoporous diatomaceous earth core, and an outer surface having aplurality of S-nitroso-N-acetyl-penicillamine groups covalently attachedthereto. The diatomaceous earth core provides an inexpensive and readilyavailable bio-derived mesoporous silica core capable of dimensions ofabout 10 μm to about 20 μm and with highly ordered pores with an averagepore size of about 300 nm to about 600 nm.

The nitric-oxide-releasing materials can provide high nitric oxidecontent (nitric oxide loading). For example, in some aspects the nitricoxide content is about 0.025 μmol to about 0.05 μmol per mg of thenitric oxide-releasing material (μmol/mg). In some aspects, the nitricoxide content is about 0.02-0.08 μmol/mg, about 0.025-0.08 μmol/mg,about 0.03-0.08 μmol/mg, about 0.04-0.08 μmol/mg, or higher.

The nitric oxide-releasing materials can provide for prolonged andsustained release for a period of days. In some aspects, the nitricoxide-releasing material has a half-life for nitric oxide release ofabout 15 hours, about 20 hours, about 25 hours, about 30 hours, or more.In some aspects, the half-life for nitric oxide release is about 15hours to 50 hours, about 20 hours to 50 hours, about 20 hours to 40hours, about 25 hours to 40 hours, or about 25 hours to 50 hours.

The nitric oxide-releasing materials can be used in a variety ofapplications. For example, in some aspects the nitric oxide-releasingmaterials are used in medical devices. For example, a surface of themedical device can include a nitric oxide-releasing materials describedherein. Medical devices can include any suitable medical device, forexample a urinary catheter, a vascular catheter, a graft, or a stent.The nitric oxide-releasing materials can also be used in wound treatmentand/or in the prevention of infections, etc. For example, in someaspects a wound gauze is provided that has an absorbent materialincluding a nitric oxide-releasing material described herein. The nitricoxide-releasing materials can also be used in acne medications, e.g. ina facial cream, facial lotion, or a medicated facial wash. In someaspects, antiseptic mouthwashes are provided containing the nitricoxide-releasing materials.

In some aspects, a nitric oxide-releasing material is provided having amesoporous diatomaceous earth core, and an outer surface having aplurality of S-nitroso-N-acetyl-penicillamine groups covalently attachedthereto. In some aspects, a nitric oxide-releasing material is providedhaving a mesoporous silica core, and an outer surface having a pluralityof S-nitrosothiols covalently attached thereto. In some aspects, anitric oxide-releasing material is provided having a mesoporous silicacore having an outer surface, a plurality of nitric oxide donors, and aplurality of amide linker groups covalently attaching the plurality ofnitric oxide donors to the outer surface. In some aspects, a nitricoxide-releasing material is provided having a mesoporous silica core,and an outer surface containing a plurality of moieties having astructure according to the following formula

In the above formula, A is a nitric oxide donor. A variety of nitricoxide donors are described below. In some aspects, A is a nitric oxidedonor described below. In some aspects, A isS-nitroso-N-acetyl-penicillamine or a derivative or fragment thereof.

In the above formula, R¹ can be none. R¹ can be a C₁-C₃₀ alkyl, C₁-C₂₀alkyl, C₂-C₂₀ alkyl, C₃-C₂₀ alkyl, C₃-C₁₅ alkyl, C₃-C₁₂ alkyl, or C₆-C₁₂alkyl. The alkyl can be substituted or unsubstituted. R¹ can be a C₁-C₃₀heteroalkyl, C₁-C₂₀ heteroalkyl, C₂-C₂₀ heteroalkyl, C₃-C₂₀ heteroalkyl,C₃-C₁₅ heteroalkyl, C₃-C₁₂ heteroalkyl, or C₆-C₁₂ heteroalkyl. Theheteroalkyl can be substituted or unsubstituted. R¹ can be a C₂-C₃₀alkenyl, C₂-C₂₀ alkenyl, C₃-C₂₀ alkenyl, C₄-C₂₀ alkenyl, C₄-C₁₅ alkenyl,C₄-C₁₂ alkenyl, or C₆-C₁₂ alkenyl. The alkenyl can be substituted orunsubstituted. R¹ can be a C₁-C₃₀ alkoxy, C₁-C₂₀ alkoxy, C₂-C₂₀ alkoxy,C₃-C₂₀ alkoxy, C₃-C₁₅ alkoxy, C₃-C₁₂ alkoxy, or C₆-C₁₂ alkoxy. Thealkoxy can be substituted or unsubstituted. R¹ can be a C₁-C₃₀heteroalkoxy, C₁-C₂₀ heteroalkoxy, C₂-C₂₀ heteroalkoxy, C₃-C₂₀heteroalkoxy, C₃-C₁₅ heteroalkoxy, C₃-C₁₂ heteroalkoxy, or C₆-C₁₂heteroalkoxy. The heteroalkoxy can be substituted or unsubstituted. Insome aspects, R1 is a substituted or unsubstituted C₁-C₁₂ alkyl or asubstituted or unsubstituted C₁-C₁₂ aminoalkyl. Suitable substituentscan include any suitable substituent. In some aspects, the substituentsinclude C₁-C₃ alkyl, C₁-C₃ heteroalkyl, C₁-C₃ alkoxy, and C₁-C₃heteroalkoxy.

In the above formula, each occurrence of R² can be selected independentof the other R² groups so long as at least one occurrence of R² is abond to an oxygen atom on the outer surface. In some aspects, exactly 1occurrence of R² is a bond to an oxygen atom on the outer surface. Insome aspects, exactly 2 occurrences of R² are a bond to an oxygen atomon the outer surface. In some aspects, every occurrence of R² is a bondto an oxygen atom on the outer surface. Each occurrence of R² can be aC₁-C₃₀ alkyl, C₁-C₂₀ alkyl, C₂-C₂₀ alkyl, C₃-C₂₀ alkyl, C₃-C₁₅ alkyl,C₃-C₁₂ alkyl, or C₆-C₁₂ alkyl. The alkyl can be substituted orunsubstituted. Each occurrence of R² can be a C₁-C₃₀ heteroalkyl, C₁-C₂₀heteroalkyl, C₂-C₂₀ heteroalkyl, C₃-C₂₀ heteroalkyl, C₃-C₁₅ heteroalkyl,C₃-C₁₂ heteroalkyl, or C₆-C₁₂ heteroalkyl. The heteroalkyl can besubstituted or unsubstituted. Each occurrence of R² can be a C₂-C₃₀alkenyl, C₂-C₂₀ alkenyl, C₃-C₂₀ alkenyl, C₄-C₂₀ alkenyl, C₄-C₁₅ alkenyl,C₄-C₁₂ alkenyl, or C₆-C₁₂ alkenyl. The alkenyl can be substituted orunsubstituted Each occurrence of R² can be a C₁-C₃₀ alkoxy, C₁-C₂₀alkoxy, C₂-C₂₀ alkoxy, C₃-C₂₀ alkoxy, C₃-C₁₅ alkoxy, C₃-C₁₂ alkoxy, orC₆-C₁₂ alkoxy. The alkoxy can be substituted or unsubstituted. Eachoccurrence of R² can be a C₁-C₃₀ heteroalkoxy, C₁-C₂₀ heteroalkoxy,C₂-C₂₀ heteroalkoxy, C₃-C₂₀ heteroalkoxy, C₃-C₁₅ heteroalkoxy, C₃-C₁₂heteroalkoxy, or C₆-C₁₂ heteroalkoxy. The heteroalkoxy can besubstituted or unsubstituted. In some aspects, each occurrence of R² isindependently a substituted or unsubstituted C₁-C₆ alkyl or a bond to anoxygen atom on the outer surface. Suitable substituents can include anysuitable substituent. In some aspects, the substituents include C₁-C₂alkyl, C₁-C₂ heteroalkyl, C₁-C₂ alkoxy, and C₁-C₂ heteroalkoxy.

Mesoporous Silica Cores

The nitric oxide-releasing materials can include a mesoporous silicacore. The use of a mesoporous silica core can provide for, among otherthings, larger and more ordered nitric oxide-releasing materials capableof controlled and sustained nitric oxide release.

In some aspects, the functionalization of the mesoporous silica coreresults in no or only a negligible change in the size and/or porosity ofthe mesoporous core. For example, in some aspects, the mesoporous coreand the nitric oxide-releasing material have an average pore size ofabout 200 nm to 2000 nm, about 300 nm to 2000 nm, about 300 nm to 1500nm, about 300 nm to 1200 nm, about 400 nm to 1200 nm, about 400 nm to1000 nm, about 400 nm to 800 nm, about 400 nm to 600 nm, or about 300 nmto 600 nm. In some aspects, the mesoporous core and the nitricoxide-releasing material have a longest dimension of about 5 μm to 50μm, about 10 μm to 50 μm, about 10 μm to 40 μm, about 15 μm to 40 μm,about 15 μm to 30 μm, about 15 μm to 20 μm, or about 10 μm to 20 μm.

In some aspects, the mesoporous silica core is a diatomaceous earth, arice husk, an SAB-3 type mesoporous silica, an HMS type mesoporoussilica, MSU-X type mesoporous silica, an SBA-12 type mesoporous silica,an SBA-15 type mesoporous silica, an SBA-16 type mesoporous silica, oran MCM-41 type mesoporous silica.

In some aspects, the mesoporous silica core is a synthetically derivedmesoporous silica. A synthetically derived mesoporous silica core can beprepared, for example, via (1) performing a hydrothermal synthesisreaction with a mixture containing tetraalkoxysilane, a predeterminedstructure-directing agent, and water to obtain a crystal (silica havinga mesoporous structure) and (2) calcining the crystal to obtainmesoporous silica. The structure-directing agent is suitably selected togenerate the desired mesoporous silica having the desired structure andpore size. For preparing SAB-3 type mesoporous silica a geminisurfactant (e.g., C_(n)H_(2n+1)(CH₃)₂N⁺(CH₂)_(S)N⁺(CH₃)₂C_(m)H_(2m+1),wherein n s, and m each represent an integer of 1 or more) can beselected as a structure-directing agent as described in CatalysisCommunications, Holland, 2008, Vol. 9, No. 13, p. 2287-2290. For HMStype mesoporous silica, long chain alkylamine (C_(n)H_(2n+1)NH₂, whereinn represents a integer of 1 or more) can be selected as astructure-directing agent as described in Applied Catalysis A: General,Holland, 2008, Vol. 347, p. 133-141. For MSU-X type mesoporous silica,oleyl decaoxyethylene can be selected as a structure-directing agent asdescribed in Microporous and Mesoporous Materials, Holland, 2008, Vol.109, p. 199-209. For SBA-12 type mesoporous silica, polyethylene oxidecan selected as a structure-directing agent as described in Journal ofPhysical Chemistry B, USA, 2002, Vol. 106, p. 3118-3123. For SBA-15 typemesoporous silica, a triblock copolymer (polyethyleneoxide-polypropylene oxide-polyethylene oxide copolymer) can be selectedas a structure-directing agent as described in Science, USA, Vol. 279,p. 548-552, and Microporous and Mesoporous Materials, Holland, 2006,Vol. 91, p. 156. For preparing SBA-16 type mesoporous silica, a triblockcopolymer (polyethylene oxide-polypropylene oxide-polyethylene oxidecopolymer) can be selected as a structure-directing agent as describedin Microporous and Mesoporous Materials, Holland, 2007, Vol. 105, p.15-23. Calcination temperatures and times can be selected by one ofskill in the art, but in some aspects will be about 500 to 600° C., andfor about 1 to 20 hours.

In some aspects, the mesoporous silica core is a biomimetic mesoporoussilica. The use of biomimetic mesoporous silica can provide for complexarchitectures from the nanoscale to the macroscale without the (oftenenergy inefficient and quite stringent) synthetic conditions requiredfor synthetically derived mesoporous silica. A biomimetic mesoporoussilica can include a biosilicate described in Current Opinions in SolidState and Materials Science, Zaremba, 1996, Vol. 1, p. 425-429 and thereferences cited therein. In some aspects, the mesoporous silica core isderived from plant species such as rice or equisetum or possiblediatomaceous origin. This includes, for example diatomaceous earth,diatoms and silicified plant material.

Nitric Oxide Donors

The nitric oxide-releasing material will include a nitric oxide donor.The nitric oxide donor can be an S-nitrosothiol. The S-nitrosothiol caninclude, for example, S-nitroso-N-acetyl-penicillamine,S-nitroso-N-acetyl cysteine, S-nitroso-N-acetyl cysteamine,S-nitrosoglutathione, methyl S-nitrosothioglycolate, or a fragment or aderivative thereof. The nitric oxide donor can be a diazeniumdiolate,for example diazeniumdiolated dibutylhexanediamine or a fragment or aderivative thereof.

Linkers

The nitric oxide donor will be covalently attached to the surface. Insome aspects, the nitric oxide donor can be directly bonded to thesurface oxygen. However, in some aspects, the nitric oxide donor iscovalently attached to the surface through a linker. A variety ofsuitable linkers can be included. In some aspects, the linker is aC₁-C₃₀ alkyl, C₁-C₂₀ alkyl, C₂-C₂₀ alkyl, C₃-C₂₀ alkyl, C₃-C₁₅ alkyl,C₃-C₁₂ alkyl, or C₆-C₁₂ alkyl. The alkyl can be substituted orunsubstituted. In some aspects, the linker is a C₁-C₃₀ heteroalkyl,C₁-C₂₀ heteroalkyl, C₂-C₂₀ heteroalkyl, C₃-C₂₀ heteroalkyl, C₃-C₁₅heteroalkyl, C₃-C₁₂ heteroalkyl, or C₆-C₁₂ heteroalkyl. The heteroalkylcan be substituted or unsubstituted. In some aspects, the heteroalkyl isan aminoalkyl. In some aspects, the linker is a C₁-C₃₀ alkoxy, C₁-C₂₀alkoxy, C₂-C₂₀ alkoxy, C₃-C₂₀ alkoxy, C₃-C₁₅ alkoxy, C₃-C₁₂ alkoxy, orC₆-C₁₂ alkoxy. The alkoxy can be substituted or unsubstituted. In someaspects, the linker is a a C₁-C₃₀ heteroalkoxy, C₁-C₂₀ heteroalkoxy,C₂-C₂₀ heteroalkoxy, C₃-C₂₀ heteroalkoxy, C₃-C₁₅ heteroalkoxy, C₃-C₁₂heteroalkoxy, or C₆-C₁₂ heteroalkoxy. The heteroalkoxy can besubstituted or unsubstituted. In some aspects, the linker is asubstituted or unsubstituted C₁-C₁₂ alkyl or a substituted orunsubstituted C₁-C₁₂ aminoalkyl. Suitable substituents can include anysuitable substituent. In some aspects, the substituents include C₁-C₁₂primary amines and C₁-C₁₂ secondary amines.

Methods of Making Nitric Oxide-Releasing Materials

Methods of making nitric oxide-releasing materials are also provided. Insome aspects, those of skill in the art will recognize other methodsbased upon the teachings herein. Such methods are not intended to beexcluded, but are rather intended to be included in some aspects of themethods described herein.

In some aspects, the methods of making a nitric oxide-releasing materialinclude (1) silylation of a surface of a mesoporous silica core with asilane having a first reactive coupling group to produce a firstfunctionalized surface; (2) coupling of a thiol having a second reactivecoupling group to form a covalent bond with the first reactive couplinggroup to produce a thiol-functionalized surface; and (3) nitrosylationof a thiol in the thiol-functionalized surface to produce the nitricoxide-releasing material. The first reactive functional group and thesecond reactive functional group can be any reactive functional grouppair described herein, so long as the first reactive functional groupand the second reactive functional group are capable of reacting to forma covalent attachment. In some aspects, the first reactive couplinggroup and the second reactive coupling group are selected from a primaryamine and an amine-reactive linking group such as isothiocyanates,isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes,glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters,carbodiimides, anhydrides, and fluorophenyl esters. In some aspects, thefirst reactive coupling group and the second reactive coupling group areselected from an aldehyde and an aldehyde reactive linking group such ashydrazides, alkoxyamines, and primary amines.

In some aspects, a method of making a nitric oxide-releasing material isprovided, the method including (1) silylation of a surface of amesoporous silica core with an aminosilane to produce anamine-functionalized surface; covalent attachment of a thiolactone to anamine in the amine-functionalized surface to produce athiol-functionalized surface; and nitrosylation of a thiol in thethiol-functionalized surface to produce the nitric oxide-releasingmaterial. In some aspects, the aminosilane has a structure according tothe following formula

where R¹ and R² are as described above. In some aspects, at least oneoccurrence of R² is a methoxy or ethoxy. In some aspects, R¹ is asubstituted or unsubstituted C₁-C₁₂ alkyl or a substituted orunsubstituted C₁-C₁₂ aminoalkyl.

In some aspects, the thiolactone has a structure according to thefollowing formula

where R⁴ is a substituted or unsubstituted C₁-C₂₀ alkyl, C₁-C₁₂ alkyl,C₂-C₁₂ alkyl, C₃-C₁₂ alkyl, C₃-C₁₅ alkyl, C₃-C₂₀ alkyl, or C₆-C₁₂ alkyl.The alkyl can be substituted or unsubstituted. In some aspects, thethiolactone has a structure according to the following formula

In the above formula, each occurrence of R⁵ can be independently ahydrogen, a hydroxyl, a substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted C₁-C₆ heteroalkyl, a substituted orunsubstituted C₂-C₆ alkenyl, a substituted or unsubstituted C₂-C₆herteroalkenyl, a substituted or unsubstituted C₁-C₆ alkoxy, or asubstituted or unsubstituted C₁-C₆ heteroalkoxy.

In some aspects, the thiolactone is N-acetyl-D-penicillamine or afragment or a derivative thereof. In some aspects, the thiolactone isN-Acetylcysteine thiolactone, N-Acetyl-homocysteine thiolactone,Homocysteine thiolactone, Butyryl-homocysteine thiolactone, or aderivative or fragment thereof.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Diatomaceous earth (DE), a nanoporous silica material made of fossilizedunicellular marine algae, possesses unique mechanical, moleculartransport, optical, and photonic properties exploited across an array ofbiomedical applications. The utility of DE in many of these applicationsstands to be enhanced through the incorporation of nitric oxide (NO)technology shown to modulate a number of essential physiologicalprocesses. In this work, the preparation and characterization of abio-templated diatomaceous earth-based nitric oxide delivery scaffold isdescribed for the very first time. Three aminosilanes((3-Aminopropyl)triethoxysilane (APTES),N-(6-Aminohexyl)aminomethyltriethoxysilane (AHAMTES), and3-Aminopropyldimethylethoxysilane (APDMES)) were evaluated for theirability to maximize NO loading via the covalent attachment ofN-acetyl-D-penicillamine (NAP) to diatomaceous earth. The use of APTEScrosslinker resulted in maximal NAP tethering to the DE surface andNAP-DE was converted to NO-releasing S-nitroso-N-acetyl-penicillamine(SNAP)-DE by nitrosation. The total NO loading of SNAP-DE as determinedby chemiluminescence was found to be 37.2±7.91 nmol NO/mg. Retention ofdiatomaceous earth's unique mesoporous morphology throughout thederivatization was confirmed by scanning electron microscopy. SNAP-DEshowed 92.95±2.6% killing efficiency against gram positive bacteria S.aureus as compared to the control. The WST-8 based cytotoxicity testingshowed no negative impact on mouse fibroblast cells demonstrating itspotential biocompatibility. The development of NO releasing diatomaceousearth, presents a unique means of delivering tunable levels of NO tomaterials across the fields of polymer chemistry, tissue engineering,drug delivery, and wound healing.

In some aspects, the nitric-oxide releasing materials are incorporatedinto a hydrogel, e.g. a hydrogel strip that can be applied to a targetarea to deliver nitric-oxide releasing material at the surface. In someaspects, a nitric-oxide releasing material is incorporated into hydrogelstrips for dental applications, e.g. for application to dental biofilms.Various bioabsorbable polymers, natural polymers, and hydrogels can beused for the hydrogel component. Pluronic hydrogels are a class of watersoluble copolymers made of ethylene oxide (PEO) and polypropylene oxide(PPO) that have been used for a variety of biomedical applicationsincluding drug delivery, tissue engineering, and wound therapies.Pluronic hydrogels with various hydrophilicity and molecular weights canbe used (e.g., F68, P123, F127) and dissolved in methanol (100 mg/mL).The hydrogel can be loaded with nitric-oxide releasing materialsdescribed herein, e.g. about 1-20% loading or about 5-15% loading byweight.

Example 1

Introduction

In this work, three primary aminosilanes (APTES, AHAMTES, APDMES) wereused to tether NAP thiolactone, a self-protected penicillaminederivative, to 10-15 μm diatomaceous earth particles. The efficienciesof both surface silylation and NAP thiolactone attachment were comparedbetween aminosilanes. Diatomaceous earth modified with APTES/NAP yieldedthe highest levels of silane and NAP attachment and was nitrosated forfurther evaluation. The chemical modification of DE and retention ofparticle morphology throughout the derivatization were verified byFourier Transform infrared spectroscopy (FTIR) and scanning electronmicroscopy (SEM), respectively. Nitric oxide release over 24 h and totalNO content were determined by chemiluminescence. Lastly, theantibacterial and non-cytotoxic properties of this bio-templatedNO-releasing diatomaceous earth silica scaffold were evaluated.

Materials and Methods

Materials

Fossil Shell Flour Diatomaceous Earth was purchased from Perma-Guard,Inc. (Bountiful, Utah). 200 proof ethanol was obtained from Decon Labs,Inc. (King of Prussia, Pa.). Toluene and methanol were purchased fromFischer Scientific (Waltham, Mass.). (3-Aminopropyl) triethoxysilane(APTES), L-cysteine hydrochloride monohydrate, 4-Dodecylbenzenesulfonicacid, Ellman's Reagent (5,5′-Dithio-bis-(2-nitrobenzoic acid), DTNB),glycine hydrochloride, 1,4,8,11-tetraazacyclotetradecane (cyclam),t-butyl nitrite, and potassium cyanide (KCN) were purchased fromSigma-Aldrich (St. Louis, Mo.). N-(6-Aminohexyl)aminomethyltriethoxysilane (AHAMTES) and3-aminopropyldimethylethoxysilane (APDMES) were purchased from Gelest,Inc. (Morrisville, Pa.). Sodium acetate was obtained from EMD Chemicals,Inc. (Gibbstown, N.J.). The bacterial strains Staphylococcus aureus(ATCC 6538) and mouse 3T3 cells (ATCC 1658) were originally purchasedfrom American Type Culture Collection (ATCC).

Preparation of N-Acetyl-D-Penicillamine (NAP) Thiolactone

Self-protected NAP thiolactone was synthesized via a slightly modifiedprotocol by Moynihan and Robert [44]. A solution of 5 g NAP in 10 mLpyridine and a separate mixture of 10 mL pyridine and 10 mL aceticanhydride were made. Both solutions were chilled in an ice bath for 1 hbefore being combined and continuously stirred for 24 hrs. Afterwards,all pyridine in the solution was removed by rotary evaporation at 60° C.to leave behind a small amount of viscous, orange material. Thismaterial was dissolved in chloroform and repeatedly washed and extractedwith 1 M HCl. The organic layer containing NAP thiolactone was thendried using anhydrous magnesium sulfate subsequently eliminated byfiltration. Chloroform was removed under vacuum at room temperature. Thecollected solid product was washed with hexanes and allowed to dryovernight at room temperature before being stored at 5° C.

SNAP Functionalized Diatomaceous Earth Derivatization

A schematic overview of the SNAP-functionalized diatomaceous earthderivatization is shown in FIG. 1. First, purified DE wasamine-functionalized via silyation with APTES, AHAMTES, or APDMES. Next,amine-functionalized particles were reacted with NAP-thiolactone tocovalently tether NAP to DE. Finally, NAP-DE was treated with t-butylnitrite under acidic conditions to form NO-releasing SNAP-DE.

Diatomaceous Earth Purification

To remove trace organic impurities from DE, an aqueous diatomaceousearth suspension was made in a beaker and sonicated. After sonication,dark impurities settled while DE remained suspended. The suspension wasdecanted into a separate beaker and the process was repeated three timesor until all sediment was eliminated. The water in the purifiedsuspension was removed by centrifugation at 3500 rpm for 3 minutes anddried under vacuum.

Surface Silylation

Silylated diatomaceous earth was prepared by refluxing purified DE withone of three aminosilanes (APTES, AHAMTES, APDMES) in toluene for 24 hin accordance with a previously reported protocol (1 g DE: 21.4 mmolaminosilane: 100 mL toluene) [30]. Primary amine-containing silanes(FIG. 2) were selected as crosslinkers because of their ability topromote the NAP-thiolactone ring opening required to tether NAP toaminosilane/DE via an amide bond [45]. After each reflux, the aminefunctionalized DE products were washed four times with toluene and twicewith ethanol before being dried in an oven at 80° C. overnight.

NAP Attachment

NAP-DE was prepared by stirring silylated diatoms with NAP-thiolactonefor 24 h in toluene (100 mg silylated DE: 80 mg NAP-thiolactone: 5 mLtoluene). Reaction products were washed twice with toluene and driedunder vacuum at room temperature for 24 h.

Nitrosation

NAP-DE was added to a solution of 10% methanol, 90% toluene along with4-Dodecylbenzenesulfonic acid (1 mL DBSA: 100 mg APTES/NAP diatoms) anda molar excess of t-butyl nitrite. The t-butyl nitrite was firstcleansed of any copper contaminants by vortexing with an equal volume of20 mM cyclam. The reaction vessel was shielded from light and agitatedfor 2 h before its contents were dried at room temperature under vacuumfor approximately 30 h.

FQ Primary Amine Quantification

The ATTO-TAG FQ test for primary amines was conducted in accordance witha previously reported protocol [46]. Stock solutions of 10 mM FQ and 10mM KCN in methanol and water, respectively, were prepared. A workingATTO-TAG FQ solution was created which consisted of 10 μL FQ stocksolution, 20 μL of KCN stock solution, 190 μL water, and 5 μL sample. Amicroplate reader (Biotek, Winooski, Vt.) recorded fluorescencemeasurements at an excitation of 480 nm and emission maxima at 590 nm.Using the ATTO-TAG FQ solution, a calibration curve of known glycinehydrochloride concentrations was created and the amine content ofsilylated diatoms was determined.

Ellman's Test for Free Sulfhydryls

Ellman's Reagent, 5,5′-Dithio-bis-(2-nitrobenzoic acid), was used toquantify the free sulfhydryl content of NAP-DE according to a previousprotocol [47]. Briefly, a DTNB stock solution (2 mM DTNB, 50 mM NaAc)was used to create a working DTNB solution consisting of 50 μL DTNBstock solution, 100 μL PBS, 840 μL H2O, and 10 μL sample. A UV-Visspectrophotometer (Thermo Scientific Genesys 10S UV-Vis) recordedabsorbance measurements at a previously reported wavelength of 412 nm.Using the DTNB working solution, a calibration curve of known L-cysteinehydrochloride monohydrate concentrations was created and the sulfhydrylcontent of NAP-DE was determined.

Fourier Transform Infrared Spectroscopy

Fourier transform infrared spectroscopy (FTIR) analysis was used toconfirm the presence, absence, and modification of various functionalgroups throughout the synthesis of SNAP-DE. FTIR spectra of translucentKBr pellets prepared using a 1:100 mass ratio of DE particles:KBr wererecorded with a Nicolet 6700 spectrometer (Thermo Electron Corporation,Madison, Wis.). For each sample, 128 scans were obtained at a resolutionof 4 cm⁻¹ over the wavenumber range of 4000-400 cm⁻¹.

Nitric Oxide Release Measurements

Measuring NO release from SNAP-DE was done in real-time viachemiluminescence using a Sievers Nitric Oxide Analyzer (NOA) model 280i(Boulder, Colo.). Samples were weighed and subsequently tested bysubmersion in 0.01M PBS containing EDTA at 37° C. inside of an amberreaction vessel to protect from light. Nitrogen gas was continuouslybubbled and swept from the vessel at a flow rate of 200 mL min-1 tocarry the NO being released to the NOA.

Scanning Electron Microscopy and Energy-Dispersive X-Ray Spectroscopy

Scanning electron microscopy (SEM, FEI Teneo, FEI Co.) was used at anaccelerating voltage of 5.00 kV to examine the morphology ofdiatomaceous earth throughout the derivatization. The SEM was equippedwith a large detector Energy-dispersive X-ray Spectroscopy (EDS, OxfordInstruments) system used for elemental analysis and mapping of modifieddiatomaceous earth.

Bacterial Inhibition Test

Bacteria possess a propensity to bind to and form biofilms on polymericsurfaces via secretion of an extracellular matrix. These biofilmseventually calcine, reducing the penetration and thus efficacy ofantimicrobial agents such as antibiotics and silver nanoparticles [21,48]. For this reason, nitric oxide, a free radical naturallybactericidal gas molecule that readily penetrates biofilms, is a potentalternative bactericidal agent.

In the current study, the antibacterial properties of SNAP-DE was testedagainst gram-positive Staphylococcus aureus (S. aureus), a commoncausative agent of blood and nosocomial infections [49-51]. Singleisolated colonies of S. aureus strains were obtained from a pre-culturedLB agar Petri dish, inoculated in 10 mL of LB medium, and incubated at37° C., 120 rpm for 14 h. To ensure that the bacteria used in this studywere in an actively dividing log phase, the optical density of theculture was measured at a wavelength of 600 nm (OD600) using a UV-visspectrophotometer (Thermo Scientific Genesys 10S UV-Vis). The bacteriawere then separated from the original media and suspended in PBS buffer.This provided the bacteria with an osmotic physiological environment andprevented bacterial proliferation. The separation of cells from themedium was achieved by centrifugation for 7 min at 3500 rpm. Thesupernatant was discarded, replaced with sterile PBS in order toeliminate traces of medium, and centrifuged for 7 min at 3500 rpm. Thesupernatant was again discarded and the cells were resuspended in PBS.

The OD600 of the cell suspension in PBS was measured, and adjusted tokeep the cell count in the range of 108-1010 colony forming units (CFUs)per mL. The SNAP-DE and unmodified DE were suspended in triplicates(n=3) in 1 mL of PBS-bacteria culture. The bacterial suspension withoutany DE exposure was taken as a positive control. Before suspension,SNAP-DE was weighed such that 0.8 micro moles of NO were released per mLof PBS/cell solution. This weight was determined by calculating thetotal NO released per mg of SNAP-DE over a 24-h period under conditionsmimicking those of the bacterial suspension. The resulting mixture wasincubated at 120 rpm and 37° C. for 24 h. After 24 h, the bacterialsuspension was gently agitated with a pipette and serially diluted (10-1to 10-5) for plating in premade LB agar Petri dishes. The Petri disheswere incubated at 37° C. for 24 h. In parallel, serial dilutions of thebacteria were prepared just before suspending the diatoms in thebacteria culture and plated in LB agar Petri dishes. This verified theconsistency of viable cell concentrations between experiments. Postincubation, the CFUs/mg were counted (formula below) to observe therelative bactericidal effect shown by the diatoms and ultimately therelationship between NO release and bactericidal activity.

${\%\mspace{14mu}{Bacterial}\mspace{14mu}{inhibition}} = \frac{\left( {{\frac{CFU}{{cm}^{2}}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{samples}} - {\frac{CFU}{{cm}^{2}}\mspace{14mu}{in}\mspace{14mu}{test}\mspace{14mu}{samples}}} \right) \times 100}{\frac{CFU}{{cm}^{2}}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{samples}}$

Formazan Based Cell Cytotoxicity Test

WST-*based cell cytotoxicity kit (CCK-8). The cell cytotoxicity kit(CCK-8) (Sigma-Aldrich) provides a standard WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliummonosodium salt] based cell viability assay. The CCK-8 test isnon-destructive in nature and is more sensitive than other tetrazoliumsalts such as MTT, XTT, WST-1 and MTS. The number of living cells isdirectly proportional to the amount of formazan dye (orange color)generated by the interaction of WST-8 with dehydrogenases in the cellsand is detected at the absorbance maxima of 450 nm.

Preparation of Leachates

The ISO 10993-5:2009 test for in vitro cytotoxicity was followed togenerate leachates from unmodified control DE and SNAP-DE (concentrationof 1 mg/mL of medium). This was done by soaking 10 mg of the sterilizedSNAP-DE in 10 mL DMEM medium in an amber color vials and incubating for24 h at 37° C. After 24 h, the extracts were kept in the refrigerator(4° C.) prior to use in the cell culture experiment.

Cell Culture

Mouse Fibroblast cells were used as representative mammalian cells todemonstrate the presence or absence of any potential cytotoxic effectthat SNAP-DE might have towards host cells. 3T3 mouse fibroblast cellline (ATCC-1658) was used and leachates were obtained from thebiomaterial in accordance with the ISO 10993 standard. A cryopreservedvial was thawed and cells were cultured in 75 cm2 T-flask containingcomplete DMEM medium with 10% fetal bovine serum (FBS). Additionally, 1%penicillin-streptomycin was added to keep the culture contaminationfree. The T-flask with cells was incubated at 37° C. in a humidifiedatmosphere with 5% CO2 over a period of 8 days to allow the formation ofa monolayer. The culture medium was replaced intermittently and cellswere checked daily for growth and absence of contamination. After theconfluence reached above 80%, the cells were detached from the T-flask(trypsinized with 0.18% trypsin and 5 mM EDTA). Finally, the cells werecounted under hemocytometer using Trypan blue (dye exclusion method).Around 5000 cell/mL were seeded in each of the wells in a cell culturegrade 96 well plate and incubated for 24 h in a humidified incubatorwith 5% CO2.

Cytotoxicity Test

The manufacture's protocol (Sigma-Aldrich) was followed to perform thecytocompatibility test using a CCK-8 kit on the mouse fibroblast cells.After 24 h of cell culture incubation in a 96 well plate, 10 μL of theleachates from control DE and SNAP-DE were added (n=7) to the cells. Thecells were allowed to respond to the leachates during a separate 24 hincubation period inside a cell culture incubator at physiologicaltemperature. After 24 h, 10 μL of the WST-8 solution was added to theresulting solution and incubated for 4 h. In these four h, thedehydrogenase enzyme from the live cells acted upon the WST-8 solutionand converted it into an orange color product, formazan, which wasmeasured at 450 nm. The relative viability (%) of the cells as aresponse to SNAP-DE leach outs was reported relative to the control(without leachate exposure) using the formula below.

${\%\mspace{14mu}{Cell}\mspace{14mu}{Viability}} = {\frac{{Absorbance}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{test}\mspace{14mu}{samples}}{{Absorbance}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{control}\mspace{14mu}{samples}} \times 100}$

Results and Discussion

Amine Quantification of Functionalized Diatomaceous Earth

The primary amine content of DE post-silyation was quantified to verifythe presence of amine-functionalized intermediates and gauge overallreaction efficiencies. Because it is well documented that non-surfacebound silanes are readily eliminated by thorough washing, it can besafely assumed that the primary amines detected by FQ belong exclusivelyto surface bound coupling agents [52-55]. ATTO-TAG FQ reacts with theprimary amines of these coupling agents to form a highly fluorescentproduct detectable to the attomole range [46]. Post-silylation primaryamine levels were found to be 1.10±0.17, 0.15±0.01, and 0.11±0.03μmol/mg for APTES, AHAMTES, and APDMES treated DE, respectively (Table1).

TABLE 1 Amine Content Thiol Content Conversion Ratio Crosslinker^(a)(μmol/mg)^(b) (μmol/mg)^(c) (%)^(d) APTES 1.10 ± 0.17 0.0312 ± 0.0062.84 AHAMTES 0.15 ± 0.01 0.0181 ± 0.003 12.1 APDMES 0.11 ± 0.03 0.0130 ±0.001 11.82 ^(a)Type of aminosilane used to functionalize the surface ofdiatomaceous earth particles. ^(b)Primary amine content of diatomaceousearth particles after silylation. ^(c)Sulfhydryl content of silylateddiatomaceous earth after NAP addition. ^(d)Ratio between sulfhydrylcontent after NAP addition and amine content before NAP addition.

Interestingly, APTES/DE showed amine levels approximately 7 times higherthan AHAMTES/DE and 10 times higher than APDMES/DE derived under thesame conditions. The likely explanation for this variance stems from thediffering molecular structures, and thus reactivities, of the threeaminosilanes. It is widely theorized that aminosilane attachmentproceeds via the primary amine catalyzed SN2 exchange reaction betweenthe ethoxy groups of silanes and the oxygens of silanols [52-54].Because of this, APTES and AHAMTES, which possess three ethoxy groupseach, are inherently more reactive than APDMES containing only a singleethoxy group. Three ethoxy moieties allow APTES and AHAMTES toself-polymerize with surface bound and free aminosilanes to formthree-dimensional amine rich surface multi-layers (FIG. 3).

While this explains the tendency for APDMES to form only low aminecontent monolayers, it fails to explain why APTES and AHAMTES, despitetheir equivalent number of ethoxy groups, result in considerablydifferent amine concentrations. Because aminosilylation relies uponintramolecular primary amine catalysis, it is essential that theterminal amines of APTES and AHAMTES be available to the sites of SN2exchange [53, 54]. While APTES is believed to form a five-memberedcyclic intermediate which places its primary amine adjacent to the siteof SN2 exchange, AHAMTES possesses a significantly longer alkyl chainwhich reduces its ability to undergo a similar intramolecular catalysis(FIG. 2) [53, 54]. This conformational difference reduces the ability ofAHAMTES to form high amine content surface coatings [54].

Sulfhydryl Quantification of Diatomaceous Earth

The thiol content of silylated diatomaceous earth after reaction withNAP-thiolactone was determined using Ellman's Reagent (DTNB). DTNBreacts with sulfhydryls to diffuse a yellow product into solution thatis quantifiable by UV-vis. Because free thiols arise only after theprimary amine-initiated ring opening of NAP-thiolactone, sulfhydrylcontent serves as a direct indicator of covalent NAP attachment. Thesulfhydryl concentrations of APTES, AHAMTES, and APDMES were found to be0.0312±0.006, 0.0181±0.003, and 0.0130±0.001 μmol/mg, respectively(Table 1).

While these results support the expectation that higher levels ofsurface bound amines result in increased NAP attachment, a directproportionality between amine content and subsequent NAP attachment wasnot observed. Specifically, while one would expect APTES/DE to possessNAP levels 7 times higher than AHAMTES/DE and 10 times higher thanAPDMES/DE (based on amine content), APTES/DE instead demonstrated 1.7and 2.4 times more NAP attachment than AHAMTES/DE and APDMES/DE,respectively. A closer examination of these results reveals that thepercentages of surface amines tethered to NAP-thiolactone were 2.84,12.1, and 11.8% for APTES, AHAMTES, and APDMES, respectively.

Because the thickness of aminosilane layers is the most meaningfuldifference between crosslinkers employed in this work, lower amineconversion ratios for APTES multilayers suggests that the deposition ofaminosilane coupling agents past monolayer thicknesses improves NAPattachment only marginally. As stated previously, it has been suggestedthat APTES routinely forms nonuniform, highly dense, interconnectedsilane networks (FIG. 3) [52-54, 56]. In such an environment,NAP-thiolactone, with its highly-substituted ring structure, likelyexperiences steric congestion toward nucleophilic attack [45].Accordingly, NAP-thiolactone ring opening is unlikely to occur withinthe interconnected silane network believed to be present on thediatomaceous earth surface. However, penetration is not impossible andalthough the aminosilane multilayers of APTES resulted in a loweroverall conversion ratio, an increase in the sheer quantity of NAPattachment was observed.

While steric hindrance explains differences in amine conversion betweendense multilayers of APTES and thin layers of AHAMTES and APDMES,relatively low conversion ratios for even thin aminosilane layerssuggest that more factors are at play in NAP-thiolactone binding thansterics alone. Similarly low amine conversions were observed by Frost etal. when tethering NO-releasing groups to amine-modified fumed silicaparticles [30]. Future work will examine explanations for this tofurther optimize reaction efficiencies. Because the goal of this workwas to develop diatomaceous earth with maximal NO-release capacity,APTES/NAP, with its high quantity of nitrosatable sulfhydryl groups, wasselected for further analysis.

Fourier Transform Infrared Spectroscopy

FTIR spectra of unmodified-DE and NAP-DE are shown in FIG. 4, andindicate successful chemical modification of diatomaceous earth. In theunmodified-DE spectrum, the vibration observed at 3321 cm⁻¹ correspondswith both the Si—OH bonds abundant on the silica surface and the —OHbonds of water physically absorbed to the silica surface. In the samespectrum, the band at 1632 cm⁻¹ is consistent with bending vibrations ofsurface bound H2O. The disappearance of the broad peak at 3321 cm⁻¹ inthe NAP-DE spectrum indicates the elimination of Si—OH groups andphysically absorbed water upon silylation with APTES and NAP attachment.Moreover, the presence of conjugated amides consistent with thestructure of NAP-DE in this spectrum is suggested by carbonyl vibrationsat 1759 cm⁻¹ and 1657 cm-1, N—H stretching at 3251 cm⁻¹ and 3197 cm⁻¹,and N—H bending at 1562 cm-1. Sp3 C—H bonds consistent with the alkylchain of APTES and methyl groups in NAP are seen in subtle vibrations at3051 cm-1, 2966 cm⁻¹, and 2916 cm⁻¹.

Nitric Oxide Content and Release Kinetics of SNAP-DE

Chemiluminescence, one of most popular means of quantifying nitric oxiderelease from materials, was used to determine the total NO content andrelease kinetics of SNAP-functionalized diatomaceous earth (SNAP-DE).Total NO release from SNAP-DE was found to be 0.0372±0.00791 μmol/mgusing alternating injections of 0.25 M copper (II) chloride and ascorbicacid. This value of NO loading is within range of the sulfhydryl levelsquantified by Ellman's Assay (0.0312±0.0061 μmol/mg), indicatingefficient nitrosation. Nitric oxide release of 0.0372±0.00791 μmol/mgattained through the covalent attachment of NO donor minimizes thechance of toxic NO levels occurring when SNAP-modified DE is substitutedfor traditional DE in applications requiring bulk quantities ofmaterial. Furthermore, because targeted NO release levels vary greatlyacross applications, the ability to fine-tune NO flux by modulating themass of SNAP-DE incorporated into materials is a tremendous asset.

Physiological conditions were chosen for NO release testing to mimic thein vivo conditions of biomedical applications and for facile comparisonwith previously reported NO-releasing particles. FIGS. 6A-6B illustratethe nitric oxide release from SNAP-DE as both an instantaneous value anda cumulative percentage of total NO loading over a 24-h period. Notably,the NO release half-lives of SNAP-DE routinely exceeded 24 h,significantly improving upon the half-lives of previously reporteddiazeniumdiolate-based silica particles (6 min-12 h) [31, 32, 34].Sustained release of moderate levels of NO offers a unique combinationof NO release kinetics and loading which expands upon the applicationsfor currently existing NO technology, particularly in the areas ofplatelet inhibition and bacteria killing [57, 58]. Because the NOrelease levels of SNAP-DE plateaued shortly after addition to thereaction chamber, a 24-h release profile was prepared by linearlyinterpolating between steady-state data recorded at the beginning andend of a 24-h period. Nitric oxide-releasing DE particles offer anaturally-based, bio-inspired, and tunable (by varying the amount ofincorporated SNAP-DE) means of incorporating NO into polymers,hydrogels, pastes, and creams.

Scanning Electron Microscopy and Energy-Dispersive X-Ray Spectroscopy

SEM images of diatomaceous earth seen in FIGS. 6A-6B illustrate theretention of particle morphology throughout the SNAP-DE derivatization.Morphological conservation is imperative to the maintenance ofdiatomaceous earth's uniqueness as an NO donor system. In the past,fumed silica has been used in the derivatization of NO-releasing silicaparticles [30, 31]. A known issue with fumed silica, however, is itsaggregation into course, irregular clusters during pyrolytic production[59]. These irregularities render particle morphology highlyunpredictable and conversion to an NO-releasing product with consistentrelease kinetics challenging [30, 31]. The diatomaceous earth used inthis work, however, is made mostly of discrete centric diatom species4-6 μm in diameter and 10-20 μm in length possessing large openings oneither end and highly-order rows of 400-500 nm pores [60]. Such anordered, porous structure would be extremely difficult to reproduce bythe synthetic means employed in previous studies and affordsdiatomaceous earth unique physical properties invaluable to the fieldsof biosensing, filtration, immunoprecipitation, microfluidics,nanofabrication, protein catalysis, and drug delivery [32, 34, 58, 60].

Virtually all previously reported NO-releasing silica particles havebeen nonporous and on the nanoscale [30-32, 34, 58]. Although mesoporousNO-releasing silica particles have been described by Soto et al., thediatomaceous earth particles highlighted herein feature systematic poreand particle structures hundreds of times larger than those reportedpreviously [34]. Because porosity and size have been shown to directlyaffect the loading efficiency, release kinetics, and degradation ratesof therapeutic materials, such a substantial divergence from previousparticle morphologies stands to broaden the application range andutility of NO-releasing silica [61]. Specifically, porous siliconmicroparticles are being explored as “mother ships” capable of carryingtherapeutic payloads such as nanoparticles, proteins, enzymes, drugs,and genes [62].

EDS spectra of both unmodified-DE and SNAP-DE, along with chemicalmapping of elemental sulfur can be seen in FIGS. 7A-7D. Because SNAP isthe only sulfur-containing material used in the synthesis of SNAP-DE,the presence of sulfur in modified DE samples serves as a directindicator of SNAP presence. The EDS spectra of SNAP-DE clearly indicatesthe appearance of an elemental sulfur peak and thus the incorporation ofSNAP into the sample. Sulfur mapping of SNAP-DE substantiates andvisually complements this finding by showing high concentrations ofelemental sulfur in the same location as SNAP-DE particles.

Bactericidal Properties of SNAP-DE

The successful development of new-age therapeutic biomaterials hinges,in large part, on their antibacterial properties. S. aureus, a majorsource of hospital acquired infections, forms a matrix on substratesurfaces and often results in biofilms resistant to antimicrobial agentssuch as antibiotics and silver nanoparticles [63-65]. Accordingly, S.aureus was selected in this study to serve as a proof of conceptevaluation of the antibacterial properties of SNAP-DE in biomaterialapplications. Unmodified DE showed a slight reduction in bacterial CFUsas compared to the control, whose bacterial reduction was enhanced tothe log scale in the presence of SNAP (FIGS. 8A-8B). The NO releasingSNAP-DE showed 92.95±2.6% bacterial reduction as compared to thepositive control sample (without DE or SNAP-DE). Because the presence ofnitric oxide-releasing moieties marks the only difference betweenSNAP-DE and positive and DE controls, bacterial inhibition isattributable to the toxic effects of NO against bacteria alone.

Nitric oxide kills bacteria via non-specific mechanisms which involvethiol and amine nitrosation in the extracellular matrices of bacteria,DNA cleavage, lipid peroxidation and tyrosine nitrosation [66].Moreover, unlike antibiotics and silver nanoparticles, bacterialresistance to NO is unlikely to develop due to the molecule's rapid andnon-specific action [6, 67, 68]. In the past, our group and others haveshown the antimicrobial effects of NO releasing biomaterials against P.aureginosa, S. aureus, E. coli, A. baumanni, S. aureus, E. coli, L.monocytogenes, and E. faecalis [5, 27, 69-72]. In many of these studies,NO donors were either blended or chemically linked to polymers whilebacterial growth and inhibition was observed on the surface. Theprevious success of NO against the aforementioned bacteria, along withthe bactericidal effects of SNAP-DE shown directly in this work, suggestthat SNAP-DE can be an efficacious and versatile biomaterial both in itsown right and as an additive to currently existing technologies. Thiswork marks the first time an NO donor has been covalently linked todiatomaceous earth to form a next-generation NO-delivery scaffold ofnatural origin.

Cytotoxic Effects of SNAP-DE

The in vitro cytotoxicity assay marks a proof of concept evaluation ofthe material's potential biocompatibility of a given biomaterial. Thecurrent study was performed per ISO standards for cytotoxicity using aWST-8 dye based CCK-8 kit (Sigma-Aldrich). Although research groups inthe past have shown the antibacterial properties of antibiotics, silvernanoparticles, and NO-releasing materials the toxic nature of thesematerials was either not tested or was found to be cytotoxic tomammalian cells [63, 65, 73-77]. Therefore, evaluating the cytotoxicityof SNAP-DE in addition to its antibacterial properties, was a majorobjective of this work. Mouse fibroblast cells were exposed to 10 μL ofSNAP-DE material (1 mg/mL). Results for the test indicated that SNAP-DEpossessed levels of fibroblast cell viability similar to those of thecontrol (cells in the cell culture well without any material). Nosignificant (n=7) differences were found in the cytotoxicity analysis inthe presence or absence of SNAP (FIG. 9). In addition to the observedviability, the medium color remained red (phenol red indicator) showingthat the metabolism of the fibroblast cells did not turn to cause anacidic pH.

These results are consistent with previous studies demonstrating thecytocompatibility of both SNAP and diatomaceous earth. Specifically,diatomaceous earth has been utilized in personal hygiene and dietaryapplications for decades without issue and SNAP has been shown to benon-cytotoxic, biocompatible, and hemocompatible both in vivo and invitro [4, 60, 78, 79], [Cu-SNAP]. Moreover, the major degradationproduct of SNAP, NAP, has been used in the safe treatment of medicalconditions at doses of 2-4 g/day [14]. A number of studies haveevaluated the toxicity of silica particles synthesized by various means,often with conflicting results [61, 80, 81]. A general consensus,however, exists that high concentrations of silanol functionalities(≡SiOH) on silica surfaces leads to increased toxicity. It is theorizedthat surface silanols compromise cell integrity by hydrogen bonding tokey membrane components and/or dissociating above pHs of 2-3 toelectrostatically interact with positively chargedtetraalkylammonium-containing phospholipids [81]. This silanolassociated toxicity stands to be minimized in SNAP-DE through highparticle porosity and the encapsulation of silanol groups with aminelayers. Porosity reduces the solid fraction of modified silica particlesand thus the number of silanols available to negatively affect cellmembranes [80-82]. Furthermore, the coverage of silaceous diatomaceousearth with aminosilanes likely forms a protective layer that limits cellaccessibility to unreacted surface silanols [82]. These properties arein contrast to previously reported and largely non-porous NO-releasingsilica particles formed by co-condensation methods that homogeneouslyincorporate silanols throughout particles [32, 34, 58].

The lack of an observed SNAP-DE toxicity toward mammalian cells andtissue allows for flexibility in testing the material beyond theconcentrations used in this study. Overall, SNAP-DE presents a tunableNO delivery vehicle with effective antibacterial and non-cytotoxicproperties suitable for graduation to in vivo animal models. A morecomprehensive study of SNAP-DE's cytotoxic properties at higherconcentrations both in vitro and in vivo will be carried out in thefuture to prove the preclinical potential of SNAP-DE.

CONCLUSION

In this work, the synthesis and characterization of bio-templatedmesoporous nitric oxide-releasing diatomaceous earth was described forthe first time. By quantifying primary amine and thiol groups present onthe surface of functionalized DE, APTES was shown to maximize NAPattachment and thus NO loading. FTIR and EDS indicated successfulmodification of DE through the appearance of functional groups and atomsconsistent with those of SNAP. SEM confirmed the retention ofdiatomaceous earth's unique morphology throughout the synthesis.Successful SNAP tethering was further demonstrated via real-timechemiluminescence measurement of NO. Lastly, SNAP-DE particles wereshown to reduce bacterial colonies without negatively affectingmammalian cells. The results of this study suggest a promising newbio-templated NO donor system which can be leveraged in applicationsthroughout the biomedical arena.

It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations, andare set forth only for a clear understanding of the principles of thedisclosure. Many variations and modifications may be made to theabove-described embodiments of the disclosure without departingsubstantially from the spirit and principles of the disclosure. All suchmodifications and variations are intended to be included herein withinthe scope of this disclosure.

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We claim:
 1. A nitric oxide-releasing material comprising: a mesoporousdiatomaceous earth core having an outer surface with an aminosilanecovalently bonded to the outer surface, wherein the aminosilane iscovalently bonded to the outer surface of the mesoporous diatomaceousearth core by a silicon atom of the aminosilane; and aS-nitroso-N-acetyl-penicillamine group covalently bonded to the aminogroup of the aminosilane, wherein the S-nitroso-N-acetyl-penicillaminegroup covalently bonded to the amino group of the aminosilane has thefollowing formula

where A is the S-nitroso-N-acetyl-penicillamine group; where R¹ is asubstituted or unsubstituted C₁-C₂₀ alkyl, a substituted orunsubstituted C₁-C₂₀ heteroalkyl, a substituted or unsubstituted C₂-C₂₀alkenyl, a substituted or unsubstituted C₂-C₂₀ herteroalkenyl, asubstituted or unsubstituted C₁-C₂₀ alkoxy, or a substituted orunsubstituted C₁-C₂₀ heteroalkoxy; where each occurrence of R² isindependently a substituted or unsubstituted C₁-C₂₀ alkyl, a substitutedor unsubstituted C₁-C₂₀ heteroalkyl, a substituted or unsubstitutedC₂-C₂₀ alkenyl, a substituted or unsubstituted C₂-C₂₀ herteroalkenyl, asubstituted or unsubstituted C₁-C₂₀ alkoxy, a substituted orunsubstituted C₁-C₂₀ heteroalkoxy, or a bond to an oxygen atom on theouter surface of the mesoporous diatomaceous earth core wherein at leastone occurrence of R² is a bond to an oxygen atom on the outer surface ofthe mesoporous diatomaceous earth core.
 2. The nitric oxide-releasingmaterial according to claim 1, wherein the nitric oxide-releasingmaterial has a nitric oxide content of about 0.025 μmol to about 0.05μmol per mg of the nitric oxide-releasing material.
 3. The nitricoxide-releasing material according to claim 1, wherein the nitricoxide-releasing material has a half-life for nitric oxide release ofabout 20 hours to about 40 hours.
 4. The nitric oxide-releasing materialaccording to claim 1, wherein the nitric oxide-releasing material has anaverage pore size of about 300 nm to about 600 nm.
 5. The nitricoxide-releasing material according to claim 1, wherein the nitricoxide-releasing material is a particle having a longest dimension ofabout 10 μm to about 20 μm.
 6. The nitric oxide-releasing materialaccording claim 1, wherein R¹ is a substituted or unsubstituted C₁-C₁₂alkyl.
 7. The nitric oxide-releasing material according claim 1, whereinthe aminosilane is 3-aminopropyl)triethoxysilane (APTES),N-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES), or3-aminopropyldimethylethoxysilane (APDMES).
 8. The nitricoxide-releasing material according claim 1, wherein the aminosilane is3-aminopropyl)triethoxysilane (APTES).
 9. A medical device having atleast one surface, wherein the surface comprises the nitricoxide-releasing material having a structure according to claim
 1. 10.The medical device according to claim 9, wherein the medical device is aurinary catheter, a vascular catheter, a graft, a stent, or a hydrogelstrip.